It is known in the art to use polymers as substrates for neuron cell cultures of the central nervous system (CNS). The most common method utilizes polylysine, a homopolymer, to coat glass and plastic surface see Banker & Cowan, Rat hippocampal neurons in dispersed cell culture, Brain Res., 126:397-425 (1977); Mattson et al., Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons, J. Neurosci., 8:2087-2100 (1988); Brewer & Cotman, Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen, Brain Res., 494:65-74 (1989)!. Polylysine is considered to be efficacious and economical.
However, it is unknown whether the cationic structures of polylysine substrates are able to promote neuron adhesion and differentiation. In fact, polylysine does not appear to mimic adhesive brain proteins that are known to stimulate differentiation. These proteins that stimulate differentiation, including laminin, pleiotrophin (HB-GAM), L1 and N-CAM, are distinguishable from polylysine because they do not contain sequences of polylysine or other basic residues. Unfortunately, the high costs of production, isolation and purification associated with these large cell-adhesion molecules cause researchers to turn to synthetic polymers to accomplish neuron adhesion and differentiation.
Copolymers of lysine and alanine (LAS) have not previously been reported as effective substrates for neurons or other cells. Other polypeptides containing the integrin binding site for fibronectin, RGD (Varani et al., 1993) have proven to be effective substrates for fibroblasts and other cells A ten-residue peptide fragment of laminin promotes neurite outgrowth (Liesi et al., 1989). An antibody to a 14 residue peptide from the L1 adhesion molecule increases neuritogenesis (Appel et al., 1995), probably through an integrin mediated receptor (Montgomery et al., 1996). Short polypeptides of lysine and alanine have been shown to bind strongly to DNA (Takeuchi et al., 1991), but this is not likely to be the mechanism by which substrate-bound LAS stimulates survival and axongenesis in neurons. LAS may be a model for naturally occurring polyamine such as spermidine, spermine and putrescine (Morrison et al., 1995). B27 medium, as well as the N2 medium of Bottenstein and Sato (1979) contains putrescine. Chu et al. (1995a) have shown that the butanediamine structure of these polyamine is required for enhanced survival of hippocampal neurons when added to the culture medium. Similar additions of any of the three of these polyamines promoted neurite elongation (Chu et al., 1994) or regeneration of injured hippocampal axons in cultures growing on poly-lysine (Chu et al., 1995a). A BLAST search of the NCBI protein data base revealed no proteins with and Ala-Lys-Ala-Lys sequence.
Therefore, there is a need in the art for a polymer substrate that is inexpensive while also having the ability to promote neuron survival and axon growth.